Libraries of genes, small molecules, proteins or peptides are nowadays widely used for identifying novel compounds of particular pharmacological or chemical properties. One of the most successful strategies for identifying ligands from large biological libraries is the phage display method, which was developed more than 25 years ago. Following the first antibody libraries, random peptide libraries based on phage display were developed. Finally, screening approaches based on the concept of phage display libraries have also been introduced for eukaryotic cells, in particular yeast, but also for cells of higher organisms.
Despite several improvements of the techniques, screening results from such random peptide libraries are still not fully satisfying. In general, all approaches are based on randomly generated nucleic acid sequences, which are translated into a peptide within an organism, such that the library, at best, covers all possible variants of a peptide of a given length. However, as randomization is carried out on the level of the encoding nucleic acid sequence, already the number of nucleic acid sequences covering all possible variants of a peptide of only eight amino acids exceeds the size of a library that can be technically handled.
Moreover, the binding affinities of a peptide distinctly depend on its three-dimensional structure. As a consequence, many targets are bound by circular but not by linear peptides of corresponding sequences. Other targets, in contrast, are exclusively bound by linear peptides. Although ordinary peptide gene libraries theoretically include appropriate loops, their complexity is simply too small to cover the theoretically required amount of sequences. To address this problem, most screenings are performed using at least two different libraries, one including peptides that have been cyclised by including defined positions with codons for cysteines at or near the N- and C-terminus of the peptide. These cysteine residues can form a disulfide bond generating a loop structure. The use of several libraries is, however, time consuming and extremely elaborate, especially as most targets are either bound by linear peptides or by circular peptides.
Therefore, novel gene libraries are needed, which cover circular and linear versions of peptides alike with a reasonably high probability.